Members of the Parvoviridae family are non-enveloped icosahedral viruses that contain a single-stranded linear DNA genome. Members of this family infect a variety of vertebrates, including humans, mice, mustelids, skunks, raccoons, cows, canines, primates, and ducks, as well as insects. Parvovirus B19, a member of the genus Erythrovirus, is pathogenic in humans. Parvovirus B19 infection produces mild, self-limiting illness in immunocompetent, hematologically normal individuals; however, it may cause serious illness in individuals with cancer such as leukemia, sickle-cell disease or other types of chronic anemia, those born with immune deficiencies, those who have received an organ transplant, or those who have human immunodeficiency virus (HIV). In such individuals, parvovirus B19 can cause severe anemia, both chronic and acute. Parvovirus B19 infection in pregnant women is also associated with hydrops fetalis due to severe fetal anemia, sometimes leading to miscarriage or stillbirth. The most common presentation of parvovirus B19 is as a childhood exanthem called fifth disease or erythema infectiosum, commonly known as slapped cheek syndrome.
The parvovirus genera that infect vertebrates (Parvovirus, Erythrovirus, Dependovirus, Ambovirus, and Bocavirus) have a viral capsid made of at least two structural proteins, VP1 and VP2. For example, the parvovirus B19 capsid consists of an 83 kDa minor structural protein, VP1, and a 58 kDa major structural protein, VP2, which are found in a ˜5% to ˜95% ratio (Ozawa et al. J. Virol. 61(8): 2627-30 (1987)). The sequences of the two proteins are co-linear, with VP2 being identical to the carboxyl-terminal region of VP1; however, VP1 comprises an additional 227 amino acids at the amino-terminus, which is known as the VP1 unique region. The capsid proteins for the other members of the parvovirus genera that infect vertebrates have a similar structural relationship, although the VP1 unique region of these other members is generally shorter.
The VP1 unique region of parvovirus B19 is known to contain epitopes recognized by the longest lasting neutralizing antibodies that are raised during natural parvovirus infection (Modrow S and Dorsch S. Pathol Biol (Paris). 50: 326-331 (2002)). Furthermore some experiments with virus-like particles show that neutralizing antibodies are only raised when the VP1 unique region is present (Young N S and Brown K E. NEJM. 350:586-597 (2004)). However, as of yet, no vaccine or practical, long lasting treatment has been developed for infection by parvoviruses that primary infect humans.
Modified VP1 capsids with mutations in the active site for phospholipase A2, located in the unique region, have been described in US2007/0286870. However, as expressly taught in paragraph 11 of this application, these changes are limited to those which do “not significantly alter the immunological performance of the vaccine (i.e., in that the immunogenic properties should not be adversely affected by the mutation).”
Human immunoglobulin-containing preparations are sometimes used to treat chronic parvovirus B19 infections in immunodeficient individuals. However, immunoglobulin injections are not sufficiently practical, long lasting, or affordable for widespread or routine prophylactic use. Excluding those with fifth disease from social interaction, e.g., at work, child care centers, or schools, is not an effective way to prevent the spread of the virus, because individuals are contagious well before they develop the signatory rash. Furthermore, not all such immunoglobulin-containing preparations (IV-Ig) are effective since they may not contain antibodies capable of neutralizing parvovirus B19. Current tests of IV-Ig lots do not discriminate between the presence of non-neutralizing and neutralizing antibody against parvovirus B19. This creates uncertainty about the potency of a lot of IV-Ig for treatment of parvovirus B19 infections. Uncertainty about anti-viral potency could result in an insufficient dose being given, necessitating multiple injections for treatment of infection or could result in a patient receiving more IV-Ig than is needed for treatment. Thus there is a need for improved methods for estimating the level of neutralizing antibody to assure that a patient is treated with an appropriate amount of IV-IG.
Further, despite what is known in the art, there remains a need for development of parvovirus vaccines and treatments, and methods of identifying individuals who may be at risk for infection. Current development and use after development of parvovirus B19 vaccines and treatments is hampered by the lack of an inexpensive, simple, and rapid means of measuring a correlate of protection. The knowledge of human and animal parvovirus infections continues to advance, as indicated by the recent discovery of human bocavirus as a disease-causing agent. Thus, the need for the development of related vaccines and serologic assays against other parvoviruses is anticipated. One of skill in the art can readily apply the teachings of this disclosure to other parvoviruses as they are discovered. Presently, the ability of a parvovirus B19 vaccine to elicit functional antibodies against the virus and the potency of therapeutic candidates can only be assessed by directly detecting neutralization of viral infection using cell-based assays which are expensive, complicated, cumbersome, and time-consuming as they require the use of reagents such as erythroid progenitor cells which must be cultured before they can be used in the assay. Such assays also display high variability of results (Wong et al. J. Clin. Virol. 35: 407-413 (2007)). Thus, there also exists a need for improved methods for assessing the functional antibody titers elicited by of such vaccines to allow effective development of such vaccines and then effective use after having been developed.